Epigenetics, the modification of gene expression without altering the underlying DNA sequence plays a crucial role in regulating brain function including memory, drug addiction, and neurodegenerative disease. Despite important breakthroughs in epigenetics in the brain, the proper tools to study epigenetic regulation in specific cellular subpopulations do not currently exist. This is due to the complex heterogeneity of the brain, which can obscure important signals that occur in specific subsets of cells To solve this problem, we propose using a tetO-regulated, HA-tagged histone H3.3. Histone H3.3 incorporates into chromatin outside of DNA replication and preferentially into actively transcribed regions. The tetracycline transactivator (tTA), which allows expression of tet-regulated transgenes, can be controlled in a cell-specific manner using cell-type specifc promoters. Therefore, the tagged histone H3.3 will be a marker of active chromatin specifically in cells of interest. In this proposal, we will use the CaMKII-tTA driver line to express this tagged histone in excitatory forebrain neurons. ChIP for the HA tag will isolat nucleosomes bound to active regions of the excitatory neuron genome, and in collaboration with the Garcia lab, the histone modifications found on these nucleosomes will be quantified by mass spectrometry. In Specific Aim 1, we will characterize CaMKII-tTA x tetO-H3.3-HA mice. Immunostaining will be used to confirm the HA-tagged histone is present exclusively in excitatory neurons and behavior of the animals will be tested fr effects of the transgene. ChIP-seq will be performed using either an HA antibody or a endogenous H3.3 antibody for comparison to isolate H3.3-HA containing nucleosomes from excitatory neurons in homecage and fear conditioned mice. This will determine the precise genomic regions bound by H3.3 in response to learning in excitatory neurons. In Specific Aim 2, we will use novel histone proteomics strategies to quantify histone modifications from whole hippocampi or isolated nucleosomes after HA immunoprecipitation in homecage and fear conditioned mice. This will determine the precise histone modifications that respond to learning at active regions in excitatory neurons. Understanding the combinatorial histone modifications that occur during memory consolidation may uncover novel therapeutic targets for diseases in which cognitive deficits occur, including schizophrenia and Alzheimer's. In addition to addressing the important question of which combinations of histone modifications change after memory, this proposal promises to provide tools that can be used by researchers in all fields that struggle with cellular heterogeneity.